Endo-β-1,4-xylanases (EC 3.2.1.8), also referred herein as xylanases, is the name given to a class of enzymes which degrade the linear polysaccharide beta-1,4-xylan into shorter oligomers, and thus breaking down hemicellulose, one of the major components of plant cell walls.
Xylanases have been used for many years in various industrial applications such as in feedstuffs, in brewing or malting, in the treatment of arabinoxylan containing raw materials like grain-based materials, e.g. in the production of biofuel or other fermentation products, including biochemicals (e.g. bio-based isoprene), and/or in the wheat gluten-starch separation industry, and methods using these xylanases, as well as compositions (such as feed additive compositions) comprising said xylanases.
A common nominator in all these applications is the extreme conditions which faces the enzyme. For example, high temperatures decrease the effective utility of the presently available xylanases under industrial conditions.
In animal feed applications, suitiable xylanase enzymes may increase the digestible energy of the biomase in animal feed. The biomass, such as including corn, wheat, and DDGS, used for animal feed comprises two fractions of arabinoxylan, namely the water un-extractable arabinoxylans (WU-AX) and the water extractable arabinoxylans (WE-AX).
Useful xylanases must have not only the capability to degrade WU-AX present in the cell walls and thereby increases the release of encapsulated nutrients, but also have the ability to reduce the digesta viscosity caused by the soluble fraction.
In addition to high bio-efficacy useful xylanases also need good product properties such as pepsin resistance, low pH stability and stability against heat processing.
Pepsin is a digestive protease excreted by the animal in the first part of the digestive system. Pepsin degrades protein which makes the protein available as a nutrient for the animal. The exogenous enzymes, i.e. enzymes added to the feed, are also proteins and they will be degraded if they are susceptible to degradation by the pepsin. This will in most cases destroy the enzyme activity. Thus, useful xylanases are resistant to pepsin degradation.
Stability against feed processing is also an important feature of a xylanase in order to be useful as a feed additive. During preparation the xylanase faces high temperature conditions for a short time (e.g. 30 sec at 90° C.) during feed processing (pelleting). However, the actual catalytic activity of the enzyme is needed at lower temperatures (e.g. ˜37° C.). Consequently, the enzyme should not be inactivated irreversibly at high temperatures, while it has to be active at relative lower temperatures.
Accordingly, the need exists for xylanase enzymes that have high bio-efficacy and good product properties, including being stable against heat processing.
The parent xylanase of the present invention is superior for solubilisation of wheat and corn fiber, both water-unextractable arabinoxylans (WU-AX) and the water extractable arabinoxylans (WE-AX). In addition to that the parent xylanase has excellent biochemical properties relevant for e.g. feed production and feed application. The variants of the present invention are all derived from such a parent xylanase and were selected by specifically looking for amino acid positions which when substituted will improve the thermostability of said variant, while keeping the inherited biochemical properties unchanged.